Antithrombin-III (hereinafter referred to as "AT-III") is a glycoprotein belonging to .alpha..sub.2 globulin, and is present in blood plasma. AT-III has a molecular weight of 65,000 to 68,000 and a protease inhibition activity. AT-III strongly inhibits coagulation activity of thrombin.
Also, AT-III possesses inhibition activities against not only thrombin but also against other coagulation factors, such as activated factor X and activated factor IX. Furthermore, it has been reported that AT-III possesses inhibition activities against plasmin and trypsin.
These inhibition activities are generally more rapid in the coexistence of heparin.
AT-III having such activities has been used for treating abnormal hypercoaglulability, specifically disseminated intravascular coagulation (DIC) and various other diseases caused by the decreased availability of AT-III.
Moreover, AT-III has an affinity for heparin, and a number of purification methods have been reported in which immobilized heparin is used based on such affinity property (e.g., JP-A-63-23896; the term "JP-A" as used herein means an "unexamined published Japanese patent application").
In addition, since AT-III is a protein which is present in blood plasma, inactivation of virus potentially contained therein is required when it is used as a therapeutic preparation.
Among these virus inactivation treatments, a liquid state heat treatment at 60.degree. C. for 10 hours is known as a specific example. However, it has been reported that AT-III is stable at pH 6 to 8, but 80% of the activity is lost by a heat treatment at 56.degree. C. for 6 hours (J. Biol. Chem., 246:3694 (1971)). On the other hand, there are many reports stating that the activity of AT-III is stably maintained when AT-III is subjected to a liquid state heat treatment at 60.degree. C. for 10 hours in the presence of 14.7 w/v % sodium citrate (Thrombosis Research, 22:233 (1981) and J. Biol. Chem., 256:12140 (1981)). However, when AT-III is heated in the liquid state even in the presence of 0.35 to 1.5 M (10.29 to 44.12 w/v %) sodium citrate, denaturation of AT-III is observed by immunoelectrophoresis analysis (Vox Sang, 48:325 (1985)).
Since such inactivation treatment of viruses also generates denatured protein such as inactivated AT-III, polymerized protein or the like as discussed above, methods have been reported for elimination of such impurities, for example, by carrying out the immobilized heparin treatment again (JP-A-63-23896) or carrying out a hydrophobic chromatography treatment (JP-A-1-275600). However, the immobilized heparin treatment results in low activity recovery yield even by a single treatment, and the hydrophobic chromatography treatment has a possibility of leaving contaminant proteins, such as prealbumin, transferrin, IgG and the like, so that it is necessary that the purification step is carried out again for removing these proteins, and as a result, the activity recovering yield is reduced.